We investigate the molecular mechanisms by which transmembrane proteins are sorted to different compartments of the endomembrane system such as endosomes, lysosomes and a group of cell-type-specific organelles known as lysosome-related organelles (e.g., melanosomes and platelet dense bodies). Sorting is mediated by recognition of signals present in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats (e.g., clathrin coats). Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA proteins, and the heteropentameric retromer complex. Proper sorting requires the function of additional components of the trafficking machinery that mediate vesicle tethering and fusion. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, and investigating human diseases that result from genetic defects of these proteins (e.g., Hermansky-Pudlak syndrome; neurodegenerative and neurodevelopmental disorders). AP-1, AP-2, and AP-3 are clathrin-associated adaptor complexes that recognize two types of sorting signal referred to as tyrosine-based and dileucine-based. Previous studies from our laboratory showed that tyrosine-based signals bind to the mu1, mu2 and mu3 subunits, whereas dileucine-based signals bind to a combination (i.e., a hemicomplex) of two subunits, gamma-sigma1, alpha-sigma2 and delta-sigma3, from the corresponding AP complexes. This past year we demonstrated that a dileucine-based sorting signal in the cytosolic tail of the &#946;-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) interacts with AP-2 to mediate rapid endocytosis and lysosomal targeting of the protein. BACE1 endocytosis, however, is dispensable for APP cleavage, supporting the notion that the pathogenic amyloid-beta peptide is generated in the late secretory pathway rather than in endosomes. In addition, in collaboration with the group of Michael Marks (University of Pennsylvannia School of Medicine), we showed that recognition of another dileucine-based sorting signal in the cytosolic tail of the oculocutaneous albinism type 2 (OCA2) protein by AP-1 and AP-3 mediates OCA2 sorting to melanosomes. A major effort of our laboratory this past year was to examine the role of signal-adaptor interactions in polarized sorting in neurons and epithelial cells. Neurons are polarized into dendrites, soma and axons. The plasma membrane of each of these domains possesses a distinct set of transmembrane proteins, including receptors, channels, transporters and adhesion molecules. We hypothesized that sorting to these domains could be mediated by interaction of sorting signals with AP complexes. Our studies showed that the cytosolic tails of various transmembrane receptors, including the transferrin receptor (TfR), the Coxsackie virus and adenovirus receptor (CAR), and the glutamate receptor proteins mGluR1, NR2A and NR2B, all have information leading to the sorting of these proteins to the somatodendritic domain of hippocampal neurons. In the case of TfR and CAR, this information occurred in the form of tyrosine-based sorting signals. Protein interaction analyses showed that the tails of these receptors bind to the mu1A subunit of AP-1. Dominant-negative interference and RNAi approaches demonstrated that interaction of cytosolic tails with AP-1 was responsible for somatodendritic sorting. Sorting involved exclusion of the receptor proteins from transport carriers destined for the axonal domain at the level of the soma. Interference with AP-1-dependent somatodendritic sorting caused defective maturation of dendritic spines and decreased the number of synapses. Recently, mutations in the sigma1A and sigma1B subunits of AP-1 were shown to be the cause of two neurodevelopmental disorders known as MEDNIK syndrome and Fried syndrome, respectively. Our findings suggest that these disorders may arise from failure to sort certain cargos to the somatodendritic domain of specific neuronal populations. Epithelial cells are also polarized into basolateral and apical domains. We collaborated with the group of Enrique Rodriguez-Boulan (Weill Cornell Medical College) to demonstrate that interaction of tyrosine-based sorting signals with the mu1A and mu1B subunits of AP-1 mediates sorting of transmembrane proteins such as TfR and CAR to the basolateral domain of polarized epithelial cells. Taken together, studies with neurons and epithelial cells indicate that the AP-1 complex is a global regulator of cell polarity.